t-PA基因克隆及pcDNA3.1t-PA真核表达质粒的构建

Cloning t-PA gene and constructing pcDNA3.1 (+)/t-PA expression vector

目的 利用基因工程技术克隆组织纤溶酶原激活物 (t PA)基因并构建一种无细胞毒性、不激活原癌基因的真核表达的pcDNA3.1t PA质粒载体。 方法 采用高效Trizol试剂快速从人肺组织中提取RNA ,RT PCR获得t PAcDNA ,扩增、纯化、回收t PA基因片段并将质粒 pcDNA3.1和t PA基因片段分别双酶切 ,前者纯化回收大片段 ,后者纯化回收 1 .9kb片段 ,再将回收的pcDNA3.1大片段与t PA基因片段 (1 .9kb)重组。对重组pcDNA3.1t PA质粒进行单酶切和双酶切鉴定 ,并测序。结果 成功地从胎儿肺组织中克隆了t PA基因 ,并构建了以pcDNA3.1为载体的真核表达质粒载体。结论 含t

Objective To clone tissue type plasminogen activator (t PA) gene and to construct a new kind of recombinant vector containing human tissue type plasminogon activator (t PA) cNDA neither cytotoxiaty nor actovating prot oncogenes.Methods The RNA was extracted from human lung with efficient Trizol reagent. The t PA cDNA was obtained from it by reverse transcription polymerase chain reaction (RT PCR) and was amplified, purified and retrieved. To digest pcDNA3.1t PA plasmid and t PA gene with dual endonuclease, we retrieved a big fragment from pcDNA3.1 and a 1.9 kb fragment from t PA gene. Recombined the big fragment and 1.9 kb fragment. The recombination pcDNA3.1t PA plasmid and t PA gene was identified and sequenced by mono endonuclease and dual endonuclease.Results We succeeded in cloing t PA gene from fetal lung and constructed a sort of eukaryon expression plasmid vector with pcDNA3.1. t PA 6.9 kb fragment was found in the recombination pcDNA3.1 (+)/t PA after digesting with mono endonuclease. Digesting the 6.9 kb fragment with dual endonuclease, we obtained two fragments .One is 5.0 kb, another is 1.9 kb. It was identified by sequencing that the length of the 6.9 kb fragment was same as that human being's.Conclusion A new eukaryon expression plasmid with t PA gene has been constructed. It establishes the basics for gene treating local thrombosis after angioplasty.